cut up: Biological Overview | References
Gene name - cut up
Cytological map position - 4C13-4C14
Function - signaling protein
Keywords - cytoskeletal protein - Hippo pathway - subunit of the cytoplasmic Dynein & of Myosin V - involved in both dynein-dependent and independent functions such as cell viability, axonal guidance, spermatid growth and individualization and regulation of spermatogonial divisions, wing and eye imaginal discs - part of a protein complex that regulates spindle orientation in neuroblasts
Symbol - ctp
FlyBase ID: FBgn0011760
Genetic map position - chrX:4,687,771-4,702,018
NCBI classification - LC8 family of cytoplasmic dynein light-chains
Cellular location - cytoplasmic and nuclear
Because of their functional polarity and elongated morphologies, microtubule-based transport of proteins and organelles is critical for normal neuronal function. The proteasome is required throughout the neuron for the highly regulated degradation of a broad set of protein targets whose functions underlie key physiological responses including synaptic plasticity and axonal degeneration. Molecularly, the relationship between proteasome transport and the transport of the targets of proteasomes is unclear. The dynein motor complex is required for the microtubule-based motility of numerous proteins and organelles in neurons. This study demonstrates that microtubule-based transport of proteasomes within the neuron utilizes a distinct dynein light chain compared to synaptic proteins. Live imaging of proteasomes and synaptic vesicle proteins in axons and synapses finds that these cargoes traffic independently and that proteasomes exhibit significantly reduced retrograde transport velocities compared to synaptic vesicle proteins. Genetic and biochemical analyses reveals that the Drosophila homologue of the LC8 dynein light chain Cut-up binds proteasomes and functions specifically during their transport. These data support the model that Cut-up functions to specify the dynein-mediated transport of neuronal proteasomes (Kreko-Pierce, 2017).
Proteasomes are large protein complexes responsible for degradation of normal short-lived ubiquitylated proteins as well as mutant, misfolded or damaged proteins. All cells require regulated protein degradation; however, nerve cells are of particular interest due to their complex compartmentalization and the requirement of protein degradation for normal neuronal function. In addition, a large number of neurological disorders are characterized by accumulations of proteinaceous aggregates, suggesting that impaired protein degradation is an important disease etiology of many neurodegenerative diseases. Because protein degradation in neurons occurs on short timescales and is highly compartment specific, neurons must possess molecular mechanisms that can precisely position proteasomes near to where they are uniquely required, but also maintain a physical separation between proteasomes and neuronal targets to preserve the efficacy of regulated protein turnover (Kreko-Pierce, 2017).
Numerous studies have demonstrated the important physiological requirement of proteasome activity throughout the many compartments of the neuron. Pre-synaptically, proteasome-dependent protein degradation is critical for synapse formation, synaptic efficacy and neurotransmitter release. Post-synaptically, proteasomes have been implicated in regulating several forms of synaptic plasticity including long-term potentiation (LTP), long-term facilitation (LTF) and long-term depression (LTD). Furthermore, acute depolarization of neurons causes a global change in ubiquitylated active zone proteins at the synapse, supporting the role of proteasomes in the rapid turnover of proteins in response to neuronal activity. Collectively, these data suggest that proteasomes function locally at pre- and post-synaptic sites where they act as an important modulators of synaptic structure, function and plasticity (Kreko-Pierce, 2017).
In addition to the synaptic compartments, there is evidence that proteasome function also plays an important role in the growth, development and regeneration of axons. Recent work on neuronal development has shown that changes in retrograde axonal transport of proteasomes are critical during the specification and growth of the axon. Studies in Drosophila provide evidence that the degeneration of axons that occurs during developmental pruning or in response to injury requires the ubiquitin-proteasome system (UPS). Consistent with these observations in flies, inhibition of the UPS in rodent models delays the axonal die-back observed during Wallerian axonal degeneration demonstrating a role for protein degradation during programmed axonal degeneration in mammals. These data provide strong evidence for the evolutionarily conserved requirement of proteasome activity within the axon under both normal and pathological conditions (Kreko-Pierce, 2017).
Despite the critical compartment-specific requirements for proteasome function in neurons, little is known about the molecular mechanisms that govern proteasome transport and their targets within neurons. The trafficking of organelles and transport vesicles within all cells is predominantly mediated by microtubule (MT)-based transport mechanisms utilizing two distinct molecular motor proteins, kinesins and cytoplasmic dyneins. Kinesins mostly mediate MT plus-end-directed transport, including anterograde axonal transport in neurons. In the human genome, 45 genes code for the kinesin superfamily, supporting a genetic basis for the large diversity of cargo-specific kinesin-based transport events. Cytoplasmic dynein mediates MT minus-end-directed transport including retrograde axonal transport. However, unlike for the kinesin motor, cytoplasmic dynein is encoded by relatively few genes leading to the hypothesis that the cargo specificity of the dynein motor complex is accomplished by the heterogeneneity of dynein complex subunits and various dynein-associated accessory proteins. The LC8 dynein light chains (DYNLL1 and DYNLL2 in mammals), have been proposed as cargo-adaptors potentially providing specificity for the minus-end-directed MT transport of vesicles and organelles. This notion was supported by studies that found LC8 to simultaneously associate with the dynein motor and with a number of cargos that undergo MT-mediated transport (Kreko-Pierce, 2017).
This study used a combination of genetics, biochemistry and in vivo imaging to compare the MT-based transport of proteasomes and synaptic proteins in Drosophila motor neurons. These analyses found that proteasomes use MT-based axonal transport in axons and that the axonal transport is qualitatively similar to that of synaptic proteins. However, quantitative analysis of proteasome trafficking reveals significant differences in the retrograde transport of proteasomes compared to that of synaptic proteins. These data suggests potential molecular differences in the dynein motor complexes utilized by these two distinct cargo types. In support of this idea, a forward genetic screen identified the cut up (ctp) gene, a Drosophila homolog of LC8, as being required specifically for the axonal transport of proteasomes but not synaptic proteins. These results provide molecular evidence that proteasomes and their targets utilize specific dynein motor components during MT-based transport in neurons (Kreko-Pierce, 2017).
This study used fluorescence time-lapse imaging and single-particle tracking in Drosophila third-instar larvae to investigate trafficking of proteasomes in motor neurons. The data demonstrate that proteasomes use fast MT-based axonal transport to traffic in Drosophila motor neurons, including within the presynaptic nerve terminal. The quantitative analyses of proteasome trafficking in axons of motor neurons revealed that the velocity of retrograde transport of proteasomes is significantly slower than the velocity of anterograde transport. This is similar to what has been observed in developing hippocampal cultures derived from mouse brains, supporting the idea of conserved transport mechanisms. Furthermore, it was found that the values for retrograde velocity and run length of proteasomes are significantly less than that of synaptic vesicle proteins, and that mutations in the Klc gene had a much stronger effect on the retrograde transport velocities of synaptobrevin than they did on proteasomes. Finally, genetic analysis of proteasome transport revealed that the Drosophila homolog of the mammalian LC8 dynein light chain, cut up (ctp), is required for retrograde axonal transport of proteasomes but is dispensable for the retrograde axonal transport of synaptic vesicle proteins. These analyses strongly support the model that proteasomes utilize a different dynein motor complex for transport to that used by other synaptic cargo. It is interesting to note that synaptotagmin has been identified in both proteome studies of proteasome-dependent protein degradation and observed in polyQ-induced protein aggregates. These data support the idea that synaptotagmin is likely degraded by the proteasome, perhaps in the synapse. Given the physiological significance of changes in the abundance of key synaptic molecules such as synaptotagmin, perhaps it is not surprising that the proteasome and its synaptic targets utilize distinct transport mechanisms. It would be predictrf that this arrangement would protect against inadvertent interactions between key substrates and the proteasomes, preserving the physiological efficacy of regulated changes in protein abundance (Kreko-Pierce, 2017).
The model that proteasomes and synaptic proteins are trafficked independently is seemingly in conflict with data from a recent study of axonal transport of proteasomes from cultured hippocampal neurons that suggested that proteasomes are co-transported with various membrane-associated cargos, including the synaptic vesicle protein synaptophysin. This study specifically addressed this possibility by simultaneously co-expressing proteasomes and synaptobrevin in the same motor neuron and analyzing transport. These analyses of co-transport revealed that only 8% of the synaptobrevin transport vesicles co-transported with proteasomes, despite the comparatively large number of proteasome particles. It was also found that in the present study that most of the proteasomes (~65%) are moving, whereas another study reported a relatively small population of mobile proteasomes (~20%) with majority of particles exhibiting a random-like, reversing motion (~80%). Furthermore, the velocities of both retrograde and anterograde proteasome transport reported previously are much slower than what was observed in the current study. It should be noted that the velocities that are reported for both anterograde and retrograde transport of proteasomes are consistent with velocities of proteasomes observed in cultured hippocampal neurons during axonal differentiation. These inconsistencies between studies may reflect differences due to different neuronal cell types or reporter expression, or differences between the in situ model and cultured primary neurons (Kreko-Pierce, 2017).
Cytoplasmic dynein is a multi-subunit motor protein responsible for the MT-based transport of a wide range of cargos. The current data suggests that the Drosophila DLC Ctp can specify the axonal transport of distinct cargoes. Consistent with this role, the mammalian LC8 (DYNLL1 and DYNLL2) family of DLCs have been shown to simultaneously associate with the dynein motor and a range of cargo proteins including active zone components and proteins involved in mRNA localization during embryogenesis. Importantly, mutations that disrupt the interaction of these proteins with LC8 have been shown to disrupt their dynein-mediated MT transport, providing a link between LC8-binding partners and MT-dependent trafficking. In addition to demonstrating that ctp was necessary for the normal axonal transport of proteasomes, biochemical evidence is provided that Ctp physically interacts with the 20S and 19S proteasome subunits. It is not clear from these co-immunoprecipitation studies if Ctp directly binds to these specific subunits or perhaps some other proteasome subunit. Structural studies have indicated that a large number of diverse cargoes bind to the same groove in the LC8/Ctp dimer and in certain cases can either compete or facilitate binding with other cargoes including the intermediate chain of dynein. Based on the current data, it would be predicted that the binding of the 26S proteasome to Ctp would favor the association of Ctp with the intermediate chain of dynein and facilitate dynein-dependent transport. Neither synaptobrevin nor synaptotagmin would be predicted to have this activity (Kreko-Pierce, 2017).
In addition to providing cargo specificity, the results also suggest that Ctp can affect the processivity of the dynein motor. First, it was observed that proteasomes have a slower retrograde velocity of transport than does synaptobrevin. In addition, the retrograde velocities and run lengths of proteasome transport are reduced, but not absent, in ctp mutants. These results suggest that Ctp association with the dynein motor alters the biochemical function of the resulting motor complex. Currently, little is known about how DLCs participate in motor processivity in any system. Previous studies have demonstrated that the processivity and activity of the dynein motor can be altered by interactions with various regulatory proteins. For example, the dynactin complex has been shown to significantly increase dynein processivity similar to what the data shown for ctp. Interestingly, several recent studies suggest that the normal interaction between dynein and the dynactin complex requires an LC8 dynein light chain (Jie, 2015; Stuchell-Brereton, 2011). In addition, mutations that disrupt Dyn2 function (a yeast homolog of LC8) also impaired the recruitment of the dynactin complex to the dynein motor complex (Stuchell-Brereton et al., 2011). Further studies will be required to determine whether Ctp alters the processivity of proteasomes due to the recruitment of the dynactin complex (Kreko-Pierce, 2017).
Studies utilizing pharmacological inhibition of proteasome activity have shown that inhibition of proteasomes results in a rapid increase in synapse function, including neurotransmitter release, suggesting that proteasomes are localized near the neurotransmitter release site. Despite the evidence supporting the presence of proteasomes within the presynaptic nerve terminal, direct evidence for this is absent. This study is the first to visualize and study proteasome trafficking in the presynaptic nerve terminal. Furthermore, the consistency in transport dynamics between axon and synapse, the colocalization with Futsch and the lack of movement in ctp mutants support the idea that synaptic transport of proteasome is MT based. Furthermore, analysis of synapse morphology in ctp mutants demonstrates that proteasomes are required within the NMJ for normal synapse growth during larval development. Recent studies aimed at studying post-synaptic proteasomes have suggested that proteasomes undergo dynamic changes in their subcellular localization in response to depolarization suggesting a role for proteasome transport during synaptic plasticity. It will be important to investigate whether ctp mutations have effects on synaptic plasticity at the Drosophila NMJ (Kreko-Pierce, 2017).
Interestingly, this study also found that the trafficking behavior of proteasomes in the synapse has some important differences from trafficking in the axons. A substantial difference was observed between these two neuronal compartments in terms of the number of stationary particles, with the number of stationary proteasomes in the synapse increasing by ~400%. Additionally, it was found that synaptic proteasomes move, on average, more slowly than proteasomes in the axons. A similar change in the trafficking between the axon and nerve terminal has also been observed for neurexin (Nrxn) proteins, which are also transported more slowly in the synapse versus axon. The combination of these changes in trafficking behavior favor a longer residence of an individual proteasome in the synapse compared to in the axon. The distribution of stationary proteasomes throughout the NMJ could facilitate the local degradation of synaptic proteins within the bouton. This behavior is in contrast to studies of the trafficking of neuropeptide-containing dense core vesicles (DCVs) within the NMJ, which have few stationary vesicles and utilize a continuous 'conveyor belt' model transport at the synapses ensuring continuous source of DCVs. These differences suggest MT-based transport within the synapse is tailored to the specific cargo and their respective functions (Kreko-Pierce, 2017).
The LC8 family of cytoplasmic dynein light-chains, which includes vertebrate LC8 (aka DYNLL1/DYNLL2) and Drosophila Cut-up (Ctp), are small highly conserved proteins that are ubiquitously expressed and essential for viability. The LC8 protein is 8 kilodaltons (kD) in size and was first identified as an accessory subunit in the dynein motor complex, within which an LC8 homodimer binds to and stabilizes a pair of dynein intermediate chains (DIC). However, the LC8 protein has since emerged as a general interaction hub with multiple dynein/motor-independent roles and binding partners. In fact the majority of LC8 protein in mammalian cells is not associated with either dynein or microtubules, and LC8 orthologs are encoded in the genomes of flowering plants that otherwise lack genes encoding heavy-chain dynein motors (Barron, 2016 and references therein).
Accumulating evidence has reinforced the idea that the primary role of LC8 in mammalian cells is to facilitate dimerization of its binding partners via LC8 self-association, a mechanism that has been termed 'molecular velcro'. LC8 can be found in association with over 40 proteins that function in diverse cellular processes, including intracellular transport, nuclear translocation, cell cycle progression, apoptosis, autophagy, and gene expression. LC8 is found in both the nucleus and cytoplasm and can interact with partners in either compartment. For example the mammalian kinase Pak1 binds and phosphorylates LC8 in the cytoplasm, which in turn enhances the ability of LC8 to interact with the BH3-only protein Bim and inhibit its pro-apoptotic activity. Accordingly, overexpression of LC8 or the phosphorylation of LC8 by Pak1 enhances survival and proliferation of breast cancer cells in culture. LC8 also binds estrogen receptor-α (ERα) and facilitates ERα nuclear translocation, which in turn recruits LC8 to the chromatin of ERα-target genes. In the cytoplasm, LC8 is also found in association with the kidney and brain expressed protein (KIBRA), which is an upstream regulator of the Hippo tumor suppressor pathway. KIBRA binding potentiates the effect of LC8 on nuclear translocation of ERα, suggesting crosstalk may occur between LC8-regulated pathways. The KIBRA-LC8 complex also interacts with Sorting Nexin-4 (Snx4) to promote dynein-driven traffic of cargo between the early and recycling endosomal compartments. Thus, LC8 has been linked to a variety of proteins in both the cytoplasm and nucleus that play important roles in signaling, membrane dynamics, and gene expression (Barron, 2016).
Drosophila Ctp differs from vertebrate LC8/DYNLL by only four conservative amino acid substitutions across its 89 amino acid length. Similar to mammalian LC8, phenotypes produced by Ctp loss in flies imply roles in multiple developmental mechanisms. Drosophila completely lacking Ctp die during embryogenesis due to excessive and widespread apoptosis. Partial loss of Ctp function causes thinned wing bristles and morphogenetic defects in wing development, as well as ovarian disorganization and female sterility. Within salivary gland cells, Ctp promotes autophagy during pupation, while in neuronal stem cells it localizes to centrosomes and influences mitotic spindle orientation and the symmetry of cell division. Testes mutant for ctp have motor-dependent defects in spermatagonial divisions as well as motor-independent defects in cyst cell differentiation. A recent study linked ctp mRNA expression to the zinc-finger transcription factor dASCIZ and showed that knockdown of either Ctp or dASCIZ reduces wing size. In sum, this diversity of effects produced by Ctp loss in different Drosophila cell types suggest that Ctp plays important yet context specific roles in vivo. However, knowledge of molecular pathways that require Ctp, and in turn underlie these developmental phenotypes associated with Ctp loss, remain poorly characterized (Barron, 2016).
This study used a genomic null allele of ctp and a validated ctp RNAi transgene to assess the role of the Ctp/LC8/DYNLL protein family in pathways that act within the developing Drosophila wing epithelium. Clones of ctp null cells are quite small relative to controls and RNAi depletion of Ctp shrinks the size of the corresponding segment of the adult wing without clear defects in mitotic progression or tissue patterning. The effect of Ctp depletion on adult wing size is primarily associated with a reduction in cell size, rather than cell division or cell number, implying a role for Ctp in supporting mechanisms that enable developmental growth. In assessing the effect of Ctp loss on multiple pathways that control wing growth, robust effects were detected on one-the Hippo pathway. The Hippo pathway is a conserved growth suppressor pathway that acts via its core kinase Warts to inhibit nuclear translocation of the coactivator Yorkie (Yki), which otherwise enters the nucleus, complexes with the DNA-binding factor Scalloped (Sd), and activates transcription of growth and survival genes. In parallel to the effect of Ctp loss on clone and wing size, Ctp loss alters expression of the classic Yki target genes bantam and thread(th)/diap-1 in wing pouch cells. Parallel genetic tests confirm a requirement for ctp in Yki-driven tissue growth in the wing or eye. Quite unexpectedly however, Ctp loss has opposing effects on bantam and diap1 transcription in wing pouch cells: bantam transcription is strongly elevated while diap1 expression is strongly decreased in cells lacking Ctp. In each case, these effects map to small segments of DNA in the ban and diap1 promoters that recruit Yki transcriptional complexes. Epistasis experiments confirm that Yki is required to activate the bantam promoter in Ctp-depleted cells, and that transgenic expression of Yki can overcome the block to diap1 transcription. In sum these data argue that Ctp supports physiologic Hippo signaling in wing disc epithelial cells, and that Ctp likely interacts with an as yet unidentified Hippo pathway protein(s) to exert inverse transcriptional effects on Yorkie-target genes. These types of inverse effects have not previously been described within the Hippo pathway, and imply that distinct subsets of genes within the Yorkie transcriptome can be simultaneously activated and repressed in developing tissues via a mechanism that involves Ctp (Barron, 2016).
This study provides evidence that the spindle matrix protein Skeletor in Drosophila interacts with the human ASCIZ (also known as ATMIN and ZNF822) ortholog, Digitor. This interaction was first detected in a yeast two-hybrid screen and subsequently confirmed by pull-down assays. The study also confirms a previously documented function of Digitor as a regulator of Dynein light chain/Cut up expression. Digitor was shown to be a nuclear protein that localizes to interband and developmental puff chromosomal regions during interphase but redistributes to the spindle region during mitosis. Its mitotic localization and physical interaction with Skeletor suggests the possibility that Digitor plays a direct role in mitotic progression as a member of the spindle matrix complex. Furthermore, a true null Digitor allele results in complete pupal lethality when homozygous, indicating that Digitor is an essential gene. Digitor plays critical roles in regulation of metamorphosis and organogenesis as well as in the DNA damage response. In the Digitor null mutant larvae there are greatly elevated levels of γH2Av, indicating accumulation of DNA double-strand breaks. Furthermore, reduced levels of Digitor decrease the resistance to paraquat-induced oxidative stress resulting in increased mortality in a stress test paradigm. It was shown that an early developmental consequence of the absence of Digitor is reduced third instar larval brain size although overall larval development appears otherwise normal at this stage. Altogether the data shows that Digitor is a nuclear protein that performs multiple roles in Drosophila larval and pupal development (Sengupta, 2016).
This study presents evidence that in Drosophila the spindle matrix protein Skeletor interacts with Digitor/dASCIZ, the human ASCIZ ortholog. This interaction was detected in a yeast two-hybrid screen and confirmed by pull-down assays. The transgenic expression of a mCitrine-labeled Digitor construct shows that Digitor/dASCIZ protein is localized to interband and developmental puffed chromosomal regions during interphase but that it redistributes during mitosis to the spindle region. RT-PCR analysis identified and characterized a P element insertion allele of Digitor/dASCIZ that appears to be a true null allele. When homozygous this allele results in a complete pupal lethal phenotype, indicating that Digitor/dASCIZ is an essential gene. This pupal lethality was partially rescued (31% viability) by the transgenic expression of 3xHA-Digitor-mCitrine in a homozygous Digitor/dASCIZ mutant background. Rescue was not complete, likely due to differences in the levels of expression of the transgene compared with wild type gene expression. Our analysis of the phenotypic consequences of the absence of Digitor/dASCIZ during development combined with its dynamic nuclear localization suggest that Digitor/dASCIZ has multiple roles in Drosophila development (Sengupta, 2016).
Previously, Zaytseva (2014) using RNAi knockdown of Digitor/dASCIZ in the posterior compartment of imaginal wing discs provided evidence that decreased Digitor/dASCIZ leads to impaired mitosis with severe spindle and chromosome alignment defects as well as to reduced wing size. These effects were mainly attributed to a decrease in Dynein light chain (Cut up) expression when levels of Digitor/dASCIZ are downregulated. However, this study showed that Digitor/dASCIZ, in addition to binding to Cut up itself, has direct physical binding interactions with the spindle matrix protein Skeletor. It has been demonstrated that the Drosophila spindle matrix protein Megator homolog Tpr in mammalian cells associates with the Dynein complex during mitosis. Furthermore, live imaging analysis indicated that Digitor/dASCIZ is confined to the spindle region during mitosis at a time after NEB when where there are no diffusion barriers. Thus, these findings suggest the possibility that Digitor/dASCIZ may be playing a direct role in mitotic progression as a member of the spindle matrix and/or Dynein complex in addition to serving as a transcriptional regulator of Dynein light chain expression (Sengupta, 2016).
The enrichment and localization of Digitor/dASCIZ to developmental puff regions suggest a role in organ maturation and metamorphosis. Furthermore, the dynamic relocalization of Digitor/dASCIZ from developmental puff regions during heat-shock conditions is compatible with the hypothesis that Digitor/dASCIZ may modulate gene expression at these sites and that this modulation changes during the stress response. An early phenotype observed in Digitor mutant crawling third instar larvae was a severely reduced brain size although overall larval development at this stage appeared normal with the size and weight of the mutant larvae indistinguishable from that of wild-type larvae. While only a small increase was detected in cell death and apoptosis in Digitor mutant brains there was a marked decrease in the number of actively dividing cells in the proliferating brain zones suggesting impaired cell proliferation was the main cause of the mutant small brain phenotype. This is in contrast to the findings of Zaytseva (2014) indicating that the underlying reason for reduced wing size after Digitor/dASCIZ RNAi knock down was anaphase arrest of mitotic cells caused by Dynein complex-dependent spindle defects leading to enhanced apoptosis. However, considering the pleiotropic effects of Digitor/dASCIZ the consequences of its absence is likely to be tissue and context dependent. For example, this study provides evidence that the actual programmed cell death of larval salivary gland cells during pupation does not occur in the absence of Digitor/dASCIZ. In the Digitor null mutant pupation is initiated; however, no eclosion occurs and pupal development is impaired and arrested at various stages. RT-PCR analysis of transcript levels of ecdysone-regulated genes controlling metamorphosis and subsequent eclosion in the ecdysone-signaling cascade showed a significant decrease in some but not all of the genes examined. It is therefore proposed that the pupal phenotypes in the Digitor mutant is a result of changes in expression of a subset of ecdysone-regulated genes as well as Mdh2 and that Digitor/dASCIZ plays a role in metamorphosis by acting as a transcriptional regulator of these genes. However, it should be noted that the experiments do not rule out that these effects are an indirect consequence of timing defects and/or Cut up dysregulation. In mammals ASCIZ also has several ATM-kinase independent developmental functions and is required for B cell maturation as well as for lung, kidney, and brain organogenesis. Some of these defects are a consequence of dynein light chain down regulation; however, in other cases the effects have been suggested to be caused by modulation of developmental signaling cascades such as the Wnt pathway (Sengupta, 2016).
Mammalian ASCIZ has a role in orchestrating ATM-kinase but not NBS1 dependent DNA damage repair in response to agents that perturb chromatin structure such as chloroquine or by osmotic and oxidative stress. This study provides direct evidence for an evolutionarily conserved role for Digitor/dASCIZ in the DNA damage response in Drosophila. In the Digitor null mutant larvae levels of γH2Av were greatly elevated indicating accumulation of DSBs in the absence of Digitor/dASCIZ. Furthermore, reduced levels of Digitor/dASCIZ decreased the resistance to paraquat-induced oxidative stress resulting in increased mortality in a stress test paradigm. Moreover, Zaytseva has shown that GFP-tagged Digitor/dASCIZ expressed in HeLa cells accumulate within 5 min along tracks of laser-induced oxidative DNA damage reminiscent of the association of human ASCIZ with oxidative DNA damage-induced foci (Sengupta, 2016).
Thus, in summary this study shows that Digitor/dASCIZ has multiple developmental functions in Drosophila and plays critical roles in regulation of metamorphosis and organogenesis as well as in the DNA damage response. In addition, evidence is provided that Digitor/dASCIZ may be contributing to mitotic progression not just as a regulator of Dynein light chain transcription but also through direct physical interactions with the spindle matrix (e.g. Skeletor). It will be of interest in future studies to further elucidate the many functional roles of Digitor/dASCIZ and their interplay in the highly amenable Drosophila model system (Sengupta, 2016).
The essential zinc finger protein ASCIZ (also known as ATMIN, ZNF822) plays critical roles during lung organogenesis and B cell development in mice, where it regulates the expression of dynein light chain (DYNLL1/LC8), but its functions in other species including invertebrates are largely unknown. This study reports the identification of the Drosophila orthologue of ASCIZ (dASCIZ) and shows that loss of dASCIZ function leads to pronounced mitotic delays with centrosome and spindle positioning defects during development, reminiscent of impaired dynein motor functions. Interestingly, similar mitotic and developmental defects were observed upon knockdown of the DYNLL/LC8-type dynein light chain Cutup (Ctp), and dASCIZ loss-of-function phenotypes could be suppressed by ectopic Ctp expression. Consistent with a genetic function of dASCIZ upstream of Ctp, it was shown that loss of dASCIZ led to reduced endogenous Ctp mRNA and protein levels and dramatically reduced Ctp-LacZ reporter gene activity in vivo, indicating that dASCIZ regulates development and mitosis as a Ctp transcription factor. It is speculated that the more severe mitotic defects in the absence of ASCIZ in flies compared to mice may be due to redundancy with a second, ASCIZ-independent, Dynll2 gene in mammals in contrast to a single Ctp gene in Drosophila. Altogether, these data demonstrate that ASCIZ is an evolutionary highly conserved transcriptional regulator of dynein light chain levels and a novel regulator of mitosis in flies (Zaytseva, 2014).
THE ATM substrate Chk2-interacting Zn2+ finger (ZnF) protein (ASCIZ; also known as ATMIN, ZNF822) was identified as a protein that forms nuclear DNA damage-induced foci specifically in response to DNA alkylating or oxidating agents, and absence of ASCIZ increases sensitivity to these base lesions. ASCIZ contains four N-terminal ZnFs and a C-terminal SQ/TQ-cluster domain (SCD) enriched in phosphorylation sites for ATM-like DNA damage response kinases. However, the SCD also functions as a potent transcription activation domain, and Asciz-deficient mouse models revealed major DNA damage-independent developmental functions as a transcription factor. Germline knockout of Asciz results in late embryonic lethality with a range of developmental abnormalities, including severe foregut separation defects with complete absence of lungs. The mRNA for dynein light-chain DYNLL1 was the most strongly downregulated transcript (~10-fold) in mouse Asciz knockout cells, and similar DYNLL1 reductions were observed in ASCIZ-deficient human and chicken cells. ASCIZ binds to the Dynll1 promoter in primary mouse cells and activates its transcription in a ZnF-dependent manner, consistent with a function as a ZnF transcription factor (Zaytseva, 2014).
DYNLL1 (LC8) was first identified as a light chain of the dynein motor complex, where it may facilitate the association of dynein intermediate chains with the heavy chains, but has since emerged as a regulator of possibly more than 100 diverse proteins (King, 2008; Barbar 2008; Rapali, 2011b). DYNLL1 is structurally highly conserved throughout evolution; for example, human DYNLL1 and its Drosophila ortholog Cutup (Ctp) differ by just four conservative substitutions within the 89-amino-acid proteins (Dick, 1996; Phillis, 1996). While null mutations of Ctp are lethal in Drosophila, and even hypomorphic mutations lead to severe morphogenesis defects (Dick, 1996; Phillis, 1996; Batlevi, 2010), Dynll1 loss-of-function mutations have not been reported in vertebrates, and it remains unclear if its regulation is as highly conserved as its structure and functions (Zaytseva, 2014).
Interestingly, beyond acting as a transcriptional activator of Dynll1 gene expression, ASCIZ itself is also a major DYNLL1-binding protein. The ASCIZ SCD contains 11 TQT motifs and 10 of these are functional DYNLL1 binding sites (Rapali, 2011a; Jurado, 2012a). Importantly, DYNLL1 binding to these sites represses transcriptional activity of ASCIZ in a concentration-dependent manner, and the dual ability of ASCIZ to activate the Dynll1 promoter and to 'sense' the concentration of its gene product, therefore, generates a feedback loop to maintain stable, free DYNLL1 protein levels (Jurado, 2012a). The extent to which impaired DYNLL1 regulation contributes to organogenesis defects in Asciz KO mice remains to be determined, but severe B cell development defects in conditional Asciz deleters can be rescued by ectopic Dynll1 expression, demonstrating that this phenotype is due to reduced DYNLL1 (Jurado, 2012b; Zaytseva, 2014 and references therein).
While its transcriptional functions as a Dynll1 regulator and its DNA base damage response functions seem to be conserved from birds to humans, no ASCIZ orthologs have previously been identified in invertebrates. This study reports the identification of the fly open reading frame CG14962 as the Drosophila melanogaster ortholog of ASCIZ (dASCIZ) and provides the first evidence that ASCIZ is essential for development in Drosophila. Strikingly, although Drosophila ASCIZ is a conserved transcriptional regulator of Dynll1/Ctp expression, this study shows that its absence leads to severe mitotic defects extending beyond the more restricted and relatively specific organogenesis and B-lymphopoiesis defects in ASCIZ KO mice, probably as a result of reduced DYNLL redundancy in flies (Zaytseva, 2014).
The mitotic and cell survival defects observed in this study for dASCIZ and Ctp RNAi knockdowns are consistent with previous reports linking Ctp to roles in embryonic cell survival and developmental apoptosis. While this study has analyzed only defects resulting from the loss of these two proteins in the developing wing, it is presumed that knockdown of dASCIZ would also mimic Ctp loss of function phenotypes in other tissues. The mitotic block/delay, with accumulation of cells from metaphase through telophase, is very similar to phenotypes reported for other dynein motor subunits in Drosophila and those observed for dynein heavy-chain knockdowns in the current system. Although Ctp and DYNLL1 have also been linked to dynein-independent roles during mitosis in Drosophila (Wang, 2011) and mammalian cells, the simplest explanation for the phenotypes observed in this study is that loss of dASCIZ or Ctp impairs cell division as a result of dynein-dependent mitotic spindle-related defects. In this sense, it is suspected that the increased incidence of cell death during wing development after dASCIZ or Ctp RNAi knockdown is a consequence of the mitotic block, rather than reflective of a role in cell survival pathways per se, as failure to complete mitosis is a well-known cause of apoptosis (Zaytseva, 2014).
A striking difference between the Drosophila RNAi phenotypes reported in this study and previously reported vertebrate RNAi or knockout phenotypes is that loss of dASCIZ causes pronounced, presumably dynein-dependent, mitotic defects, whereas developmental phenotypes in Asciz KO mice seem to be more restricted to relatively specific organogenesis and B-lymphopoiesis defects without overt mitotic deficits. A possible explanation could be that Drosophila contains a single Ctp gene, whereas mammals contain two DYNLL-encoding genes, of which only Dynll1 is regulated by ASCIZ. Thus, DYNLL2, which is normally expressed at much lower levels than DYNLL1 and not affected by loss of ASCIZ, may provide a buffer for the mitotic or other dynein-related functions of DYNLL1 in ASCIZ-deficient mice. Nevertheless, it would be conceivable that loss of DYNLL1 may contribute to aspects of the Asciz KO phenotype through other, more subtle, effects on mitotic spindle functions. For example, proposed roles for the dynein complex in realigning mitotic spindles and cell polarity for asymmetric cell divisions in Drosophila neuroblasts or mammalian cells might provide a paradigm for the lack of lung buds in Asciz KO mice: Maybe respiratory precursors, which are present in these mice, need to be rotated in some manner by dynein motors or another DYNLL1-specific effector for lung buds to emerge from the ventral foregut endoderm? While the answer to such questions is beyond the scope of the present study, the current findings provide a solid basis for future studies to determine the extent to which ASCIZ via DYNLL1 regulates mitotic and dynein motor functions in mammalian cells, as well as for future investigations into developmental functions of the dASCIZ-DYNLL1/Ctp axis in the experimentally more amenable Drosophila model system (Zaytseva, 2014).
Centrioles play a key role in nucleating polarized microtubule networks. In actively dividing cells, centrioles establish the bipolar mitotic spindle and are essential for genomic stability. Drosophila Anastral spindle-2 (Ana2) is a conserved centriole duplication factor. While recent work demonstrated that an Ana2-dynein light chain (LC8) centriolar complex is critical for proper spindle positioning in neuroblasts, how Ana2 and LC8 interact is yet to be established. This study examined the Ana2-LC8 interaction and mapped two LC8-binding sites within Ana2's central region, Ana2M (residues 156-251). Ana2 LC8-binding site 1 contains a signature TQT motif and robustly binds LC8 (KD of 1.1 mμM) while site 2 contains a TQC motif and binds LC8 with lower affinity (KD of 13 mμM). Both LC8-binding sites flank a predicted ~34-residue alpha-helix. Two independent atomic structures are presented of LC8 dimers in complex with Ana2 LC8-binding site 1 and site 2 peptides. The Ana2 peptides form beta-strands that extend a central composite LC8 beta-sandwich. LC8 recognizes the signature TQT motif in Ana2's first LC8 binding site, forming extensive van der Waals contacts and hydrogen bonding with the peptide, while the Ana2 site 2 TQC motif forms a uniquely extended beta-strand, not observed in other dynein light chain-target complexes. Size-exclusion chromatography coupled with multi-angle static light scattering demonstrates that LC8 dimers bind Ana2M sites and induce Ana2 tetramerization, yielding an Ana2M4-LC88 complex. LC8-mediated Ana2 oligomerization likely enhances Ana2's avidity for centriole binding factors and may bridge multiple factors as required during spindle positioning and centriole biogenesis (Slevin, 2014).
Ana2 is an integral component of the centriole duplication pathway, but how it works with Sas-6 and Sas-4 and whether LC8 plays a role in this pathway remain to be determined. The Sas-6 dimer interactions that facilitate cartwheel formation are very weak (KD of >100 μm), making it unlikely that Sas-6 could spontaneously build cartwheels in a cellular context at endogenous levels. Additionally, Sas-6 overexpression promotes centriole amplification only when Ana2 is co-overexpressed, suggesting that Ana2 plays a supporting role in enhancing Sas-6 oligomerization and cartwheel formation. One mechanism by which Ana2 could promote Sas-6 oligomerization would be if Ana2 itself were oligomeric. This idea is supported by recent evidence that Ana2 binds LC8, a dynein light chain that plays a ubiquitous role as a dimerization machine (Wang, 2011). This work provides insight into the Ana2-LC8 quaternary structure and establishes a foundation upon which the Ana2 tetramer's avidity effects on Sas-6 oligomerization can be investigated (Slevin, 2014).
This study has identified two LC8 binding sites in Ana2, conserved within the Drosophila genus, that flank a central domain with predicted helical structure. Although the exact binding sites are not apparent in other metazoan species, the presence of a central predicted coiled-coil is conserved across Ana2 orthologs from C. elegans Sas-5 to human STIL and suggests a role in oligomerization. This is supported by a report that the C. elegans Sas-5 N-terminal region (containing the central predicted coiled-coil) forms a tetramer in solution. Although Sas-5 tetramerization in vitro is not LC8-dependent, its oligomeric state parallels the LC8-dependent tetramerization that was observed with Ana2 (Slevin, 2014).
Dynein light chains often bind targets proximal to an endogenous oligomerization domain, potentiating target dimerization. Both of the Ana2 LC8-binding sites are an amalgam of the canonical K-3X-2T-1Q0T1 and G-2I-1Q0V1D2 LC8-binding motifs. Using ITC, this study has shown that Ana2 pep1 binds LC8 with micromolar affinity (KD = 1.1 μm). Crystal structure of LC8 bound to Ana2 pep1 shows an LC82-Ana2 pep12 binding mode, with the Ana2 pep1 canonical TQT sequence contributing key binding determinants (Slevin, 2014).
The second identified LC8-binding site (Ana2 site 2, pep2) flanks the central helical domain's C-terminal region and is composed of the sequence T-3G-2T-1Q0C1D2. Ana2 pep2 binds LC8 with lower affinity (KD = 13 μm) than Ana2 pep1. The crystal structure of LC8 bound to Ana2 pep2 also has an LC82-Ana2 pep22 binding mode. Interestingly, Ana2 pep2 adopts a unique architecture when bound to LC8 that contrasts with other LC8-peptide structures. The Ana2 pep2 Cys244 at position +1 is positioned deeper into the LC8 binding groove. The affinities reported for the LC8-Ana2 peptide interactions probably underestimate the stability of the biological complex involving full-length Ana2 and LC8. Because the solution studies support interactions between LC8 homodimers and a tetrameric Ana2M region, it is anticipated that avidity effects will increase the complex's stability beyond the affinities reported for LC8 and Ana2 pep1 and pep2. This is consistent with the finding that a stable Ana2-LC8 complex can be extracted from Drosophila cell lysate. It is noted that within the genus Drosophila, the two segments that bridge the predicted central coiled-coil with the two flanking LC8 binding sites are not conserved in sequence or length. It is predicted that these segments serve as general spacers that link the LC8 binding sites to the Ana2 coiled-coil oligomerization domain and maintain a general length that enables LC8 homodimers to bind and potentiate Ana2 oligomerization without sterically compromising coiled-coil formation (Slevin, 2014).
The data support a model in which LC8 stabilizes an Ana2 tetramer. An Ana2 tetramer may spatially arrange its conserved C-terminal STAN motifs to interact with Sas-6 and promote the Sas-6 oligomerization that underlies centriole cartwheel formation. The SEC-MALS analysis of the Ana2M-LC8 complex reveals a stable, single-species complex consisting of four Ana2M molecules and eight LC8 molecules (Ana2M4-LC88). This stable complex was purified over two successive sizing columns, demonstrating its ready formation, and yielded a similar experimental mass in two independent purifications and SEC-MALS assays. Mutating the first Ana2M LC8 binding site as well as adding a SNAP tag to LC8 supported the Ana2M4-LC88 stoichiometry (Slevin, 2014).
The Ana2-LC8 interactions that were characterized in this study raise important questions about the role of Ana2 in centriole duplication. Previous work has shown that the C-terminal half of Ana2 binds the N terminus of Sas-6 in Drosophila, implicating a possible role for the conserved STAN domain of Ana2 in Sas-6 binding. In this model, LC8 binds and stabilizes an Ana2 tetramer that may structurally organize four trans STAN domains at one end of a parallel tetramerization domain or two trans STAN domains at either end of an antiparallel tetramerization domain. In either configuration, the oligomeric state of Ana2, coupled with its ability to bind Sas-6, is predicted to enhance Sas-6 oligomerization and cartwheel formation. This correlates with cellular studies in which Sas-6 and Ana2 dual overexpression was required for cartwheel formation, suggesting that Ana2 potentiates Sas-6 cartwheel formation, potentially through oligomerization. Recent cryotomographic studies of nascent centriole architecture reveal auxiliary protein density connecting the Sas-6-based cartwheel to Sas-4 and the distal microtubule triplets. Given the integral role of Ana2 in Sas-6 cartwheel formation as well as evidence that it binds both Sas-6 and Sas-4, Ana2 is a likely candidate for this density. More work is needed to determine if Ana2 can bridge Sas-6 and Sas-4 and whether the LC8-Ana2 interaction plays a role in this Ana2 function, as it does in neuroblast asymmetric cell division. This work outlines the structural basis of the LC8-Ana2 interaction, with implications for its role in Ana2 structure and function at the centriole (Slevin, 2014).
Drosophila neural stem cells, larval brain neuroblasts (NBs), align their mitotic spindles along the apical/basal axis during asymmetric cell division (ACD) to maintain the balance of self-renewal and differentiation. This study identified a protein complex composed of the tumor suppressor anastral spindle 2 (Ana2), a dynein light-chain protein Cut up (Ctp), and Mushroom body defect (Mud), which regulates mitotic spindle orientation. Two ana2 alleles were isolated that displayed spindle misorientation and NB overgrowth phenotypes in larval brains. The centriolar protein Ana2 anchors Ctp to centrioles during ACD. The centriolar localization of Ctp is important for spindle orientation. Ana2 and Ctp localize Mud to the centrosomes and cell cortex and facilitate/maintain the association of Mud with Pins at the apical cortex. These findings reveal that the centrosomal proteins Ana2 and Ctp regulate Mud function to orient the mitotic spindle during NB asymmetric division (Wang, 2011).
The Drosophila larval brain neural stem cell, or neuroblast (NB), has recently emerged as a new model for studying stem cell self-renewal and tumorigenesis. NBs divide asymmetrically to generate a self-renewing daughter NB and a ganglion mother cell (GMC) that is committed to differentiation. Asymmetric localization/segregation machinery ensures the polarized distribution of 'proliferation factors,' including atypical protein kinase C (aPKC), and 'differentiation factors,' including basal proteins such as Numb, Miranda (Mira), Brain tumor (Brat), and Prospero, to the daughter NB and GMC, respectively. The failure of asymmetric division of NBs can result in their hyperproliferation and the induction of tumors (Wang, 2011).
To ensure the correct asymmetric segregation of cell fate determinants, the mitotic spindle has to be properly oriented with respect to the polarized proteins on the cell cortex. Inscuteable (Insc) and the heterotrimeric G proteins Gαi and Gβγ and their regulators Partner of Insc (Pins) and Ric-8 control mitotic spindle orientation (Wang, 2011).
Recent work has also implicated centrosome-associated proteins in the regulation of spindle orientation and tumorigenesis. Centrosomes function as major microtubule-organizing centers in most animal cells. A centrosome is composed of a pair of centrioles surrounded by an amorphous matrix of pericentriolar material (PCM). Centriole duplication is regulated by centriolar components, such as Asterless (Asl), Sas6, Sas4, and anastral spindle 2 (Ana2). ana2 was identified from genome-wide RNA interference (RNAi) screens, where ana2 RNAi-treated S2 cells exhibited an anastral spindle phenotype. The Ana2 overexpression phenotype and its interaction with Sas6 have suggested a role for Ana2 in centriole duplication (Stevens, 2010). However, no ana2 mutants were previously available for further functional studies (Wang, 2011).
This study has isolated two ana2 alleles that are defective in apical/basal spindle orientation during NB asymmetric division. Ana2 is demonstrated to be a tumor suppressor that suppresses NB overproliferation. The centriolar protein Ana2 directly interacts with Ctp, a dynein light chain that also localizes to the centrioles, and Mud, leading to their localization to the centrosomes. This finding suggests that the tumor suppressor Ana2 ultimately regulates Mud function to direct asymmetric division and prevent tumor formation (Wang, 2011).
This study investigated the role of Drosophila Ana2 during NB asymmetric cell division, focusing on mitotic spindle orientation. Two ana2− alleles were isolated from a genetic screen that produced supernumerary NBs in larval brains and failed to properly align the mitotic spindle with asymmetrically localized proteins. It was demonstrated that Drosophila Ana2 functioned as a tumor suppressor in a transplantation experiment. Using ana2 mutants, it was shown that Ana2 is important for centriole function. Ana2 interacts with Sas-6 through the C-terminal region of Ana2 (201-420 aa), which contains the conserved STAN motif and coiled-coil domain (Stevens, 2010). The data suggest that the N terminus of Ana2 (1-274 aa), which interacted with Ctp, a Ddlc1 (Drosophila Dynein light chain), is sufficient for its function in centriole assembly and spindle orientation. This is not in direct contradiction with the interaction between Ana2 and Sas6 because the C-terminal region of Ana2 (201-420 aa), which interacts with Sas-6, partially overlaps with the Ana2 N1 (1-274 aa). However, this result suggests surprisingly that the STAN motif may be dispensable for Ana2's function during centriole formation. The mammalian Ana2-related protein STIL, which also contains the STAN motif, has been implicated in primary microcephaly, a neurodevelopmental disorder characterized by a reduced brain size. The apparently disparate phenotypes reported for mammalian STIL and fly Ana2 during brain development are likely due to different developmental contexts (Wang, 2011).
The reason that NB overproliferation occurs in ana2 mutants, but not in asterless or sas4 mutants with spindle or centriole defects, may be due to the different behaviors of these mutants in 'telophase rescue,' a phenomenon whereby proteins delocalized from the cortex during early mitosis are restored at anaphase/telophase by a poorly understood compensatory mechanism. The spindle misorientation phenotype in ana2 mutants is much more severe than sas4 or asterless mutants. Likely as a consequence of a relatively weak spindle misorientation phenotype, 'telophase rescue' still occurred in 100% of the asterless and sas4 mutant telophase NBs, and all asymmetrically localized proteins were correctly segregated into different daughter cells. In contrast, in ana2 mutants or mud mutants, which have NB overgrowth in larval brains, asymmetrically localized proteins sometimes mis-segregate into different daughters at telophase (Wang, 2011).
The RNAi screen identified Ctp as an important player in mitotic spindle orientation because ctp mutants displayed spindle misorientation during NB asymmetric division. ctp null mutants display spindle misorientation in NBs similar to that seen in ctp RNAi. It is noted that Ctp localizes to centrioles in Drosophila. Ana2 directly binds and anchors Ctp to the centrioles during NB division. The centriole localization is important for Ctp function during spindle orientation because membrane-targeted CtpCAAX fails to rescue the spindle misorientation phenotype in the ctp null mutant. The interaction between Ctp and Ana2 on the centrioles may be critical for dynein to organize astral microtubules and move its cargo proteins along the microtubules (Wang, 2011).
A dynein component, Ctp, can also bind directly to Mud, a protein downstream of heterotrimeric G protein signaling, that regulates spindle orientation. This interaction is conserved in vertebrates; Xenopus NuMA, a Mud-related protein, also forms a complex with dynein. Ana2 and Ctp are important for spindle pole localization of Mud during spindle orientation in NBs, whereas Mud is not required for centriolar localization of Ana2 or Ctp. Ana2 also directly interacts with Mud. These data suggested that Mud may be an important downstream target of Ana2 and Ctp during spindle orientation. Ana2, Ctp, and Mud are also found in the same protein complex in vivo and in vitro. Mud is involved in spindle pole/centrosome engagement, which has not been reported in previous analyses of Mud function. Ana2 and Ctp also played a similar role during spindle pole/centrosome attachment. Together, these data indicate that the Ana2, Ctp, and Mud complex functioned to regulate spindle pole assembly and spindle orientation during asymmetric division of NBs (Wang, 2011).
Apical/basal spindle orientation is controlled by a two-step mechanism: an early, centrosome-dependent alignment and a later spindle-cortex interaction. The data indicate that Ana2 is not only critical for the early, centrosome-dependent step, but also for the later spindle-cortex interaction. Although the loss of Ana2 or Ctp function does not affect Pins asymmetric localization in NBs, Ana2 and Ctp appear to be important for the interaction between Pins and Mud in larval brains because the Pins-Mud interaction is diminished in ana2 or insc-CtpCAAX, ctp− mutant larval brains. These findings suggested that the Dynein-Dynactin complex cooperate with the centriolar protein Ana2 to mediate the spindle-cortex interaction. The spindle-cortex interaction may require the 'search and capture' mechanism, driven by the plus-end microtubule-binding protein EB1 and Dynein-Dynactin complex). It is speculated that Ana2 and Ctp may be involved in such a 'search and capture' mechanism during apicobasal spindle orientation (Wang, 2011).
These data suggested that a multiprotein complex composed of Ana2, Ctp, and Mud is critical during the regulation of spindle orientation. Ana2 and Ctp regulated Mud localization on centrosome/spindle poles as well as on the cell cortex, whereas the heterotrimeric G protein pathway is only important for cortical Mud localization. Thus, the centrosomal Ana2/Ctp/Mud complex converges with the heterotrimeric G protein pathway during spindle orientation. Very little is known about the molecular mechanisms by which centrosomal proteins regulate spindle orientation. Aur-A, a PCM protein, has been shown to phosphorylate Pins on S436 of the Pins Linker domain, which is required for accurate spindle orientation. The current findings suggest important functional links among the centriolar protein Ana2, the dynein complex, and Mud during asymmetric division of NBs. This raises the possibility that a similar mechanism whereby centrosomal proteins interact with dynein complexes to mediate cortical protein localization may exist during asymmetric division and stem cell self-renewal in mammals (Wang, 2011).
Stem cell progeny often undergo transit amplifying divisions before differentiation. In Drosophila, a spermatogonial precursor divides four times within an enclosure formed by two somatic-origin cyst cells, before differentiating into spermatocytes. Although germline and cyst cell-intrinsic factors are known to regulate these divisions, the mechanistic details are unclear. This study shows that loss of dynein-light-chain-1 (DDLC1/LC8) in the cyst cells eliminates bag-of-marbles (bam) expression in spermatogonia, causing gonial cell hyperplasia in Drosophila testis. The phenotype is dominantly enhanced by Dhc64C (cytoplasmic Dynein) and didum (Myosin V) loss-of-function alleles. Loss of DDLC1 or Myosin V in the cyst cells also affects their differentiation. Furthermore, cyst cell-specific loss of ddlc1 disrupts Armadillo, DE-cadherin and Integrin-betaPS localizations in the cyst. Together, these results suggest that Dynein and Myosin V activities, and independent DDLC1 functions in the cyst cells organize the somatic microenvironment that regulates spermatogonial proliferation and differentiation (Joti, 2011).
Autophagy is a catabolic pathway that is important for turnover of long-lived proteins and organelles, and has been implicated in cell survival, tumor progression, protection from infection, neurodegeneration, and cell death. Autophagy and caspases are required for type II autophagic cell death of Drosophila larval salivary glands during development, but the mechanisms that regulate these degradation pathways are not understood. This study conducted a forward genetic screen for genes that are required for salivary gland cell death, and Drosophila dynein light chain 1 (ddlc1) was identified as identified as a gene that is required for type II cell death. Autophagy is attenuated in ddlc1 mutants, but caspases are active in these cells. ddlc1 mutant salivary glands develop large fibrillar protein inclusions that stain positive for amyloid-specific dyes and ubiquitin. Ectopic expression of Atg1 is sufficient to induce autophagy, clear protein inclusions, and rescue degradation of ddlc1 mutant salivary glands. Furthermore, ddlc1 mutant larvae have decreased motility, and mutations in ddlc1 enhance the impairment of motility that is observed in a Drosophila model of neurodegenerative disease. Significantly, this decrease in larval motility is associated with decreased clearance of protein with polyglutamine expansion, the accumulation of p62 in neurons and muscles, and fewer synaptic boutons. These results indicate that DDLC1 is required for protein clearance by autophagy that is associated with autophagic cell death and neurodegeneration (Batlevi, 2010).
In many cell types, polarized transport directs the movement of mRNAs and proteins from their site of synthesis to their site of action, thus conferring cell polarity. The cytoplasmic dynein microtubule motor complex is involved in this process. In Drosophila, the Egalitarian (Egl) and Bicaudal-D (BicD) proteins are also essential for the transport of macromolecules to the oocyte and to the apical surface of the blastoderm embryo. Hence, Egl and BicD, which have been shown to associate, may be part of a conserved core localization machinery in Drosophila, although a direct association between these molecules and the dynein motor complex has not been shown. This study reports that Egl interacts directly with Drosophila dynein light chain (Dlc), a microtubule motor component, through an Egl domain distinct from that which binds BicD. It is proposed that the Egl-BicD complex is loaded through Dlc onto the dynein motor complex thereby facilitating transport of cargo. Consistent with this model, point mutations that specifically disrupt Egl-Dlc association also disrupt microtubule-dependant trafficking both to and within the oocyte, resulting in a loss of oocyte fate maintenance and polarity. These data provide a direct link between a molecule necessary for oocyte specification and the microtubule motor complex, and supports the hypothesis that microtubule-mediated transport is important for preserving oocyte fate (Navarro, 2004).
Mutations in the genes for components of the dynein-dynactin complex disrupt axon path finding and synaptogenesis during metamorphosis in the Drosophila central nervous system. In order to better understand the functions of this retrograde motor in nervous system assembly, the path finding and arborization of sensory axons during metamorphosis was analyzed in wild-type and mutant backgrounds. In wild-type specimens the sensory axons first reach the CNS 6-12 h after puparium formation and elaborate their terminal arborizations over the next 48 h. In Glued1 and Cytoplasmic dynein light chain mutants, proprioceptive and tactile axons arrive at the CNS on time but exhibit defects in terminal arborizations that increase in severity up to 48 h after puparium formation. The results show that axon growth occurs on schedule in these mutants but the final process of terminal branching, synaptogenesis, and stabilization of these sensory axons requires the dynein-dynactin complex. Since this complex functions as a retrograde motor, it is suggested that a retrograde signal needs to be transported to the nucleus for the proper termination of some sensory neurons (Murphey, 1999).
The molecular and genetic characterization of the cytoplasmic dynein light-chain gene, ddlc1, from Drosophila melanogaster is reported. ddlc1 encodes the first cytoplasmic dynein light chain identified, and its genetic analysis represents the first in vivo characterization of cytoplasmic dynein function in higher eukaryotes. The ddlc1 gene maps to 4E1-2 and encodes an 89-amino-acid polypeptide with a high similarity to the axonemal 8-kDa outer-arm dynein light chain from Chlamydomonas flagella. Developmental Northern (RNA) blot analysis and ovary and embryo RNA in situ hybridizations indicate that the ddlc1 gene is expressed ubiquitously. Anti-DDLC1 antibody analyses show that the DDLC1 protein is localized in the cytoplasm. P-element-induced partial-loss-of-function mutations cause pleiotropic morphogenetic defects in bristle and wing development, as well as in oogenesis, and hence result in female sterility. The morphological abnormalities found in the ovaries are always associated with a loss of cellular shape and structure, as visualized by a disorganization of the actin cytoskeleton. Total-loss-of-function mutations cause lethality. A large proportion of mutant animals degenerate during embryogenesis, and the dying cells show morphological changes characteristic of apoptosis, namely, cell and nuclear condensation and fragmentation, as well as DNA degradation. Cloning of the human homolog of the ddlc1 gene, hdlc1, demonstrates that the dynein light-chain 1 is highly conserved in flies and humans. Northern blot analysis and epitope tagging show that the hdlc1 gene is ubiquitously expressed and that the human dynein light chain 1 is localized in the cytoplasm. hdlc1 maps to 14q24 (Dick, 1996).
Mutations in an 8 kDa (8x10(3) Mr) cytoplasmic dynein light chain disrupt sensory axon trajectories in the imaginal nervous system of Drosophila. Weak alleles are behaviorally mutant, female-sterile and exhibit bristle thinning and bristle loss. Null alleles are lethal in late pupal stages and alter neuronal anatomy within the imaginal CNS. P[Gal4] inserts were used to examine the axon projections of stretch receptor neurons and an engrailed-lacZ construct was used to characterize the anatomy of tactile neurons. In mutant animals both types of sensory neurons exhibit altered axon trajectories within the CNS, suggesting a defect in axon pathfinding. However, the alterations in axon trajectory did not prevent these axons from reaching their normal termination regions. In the alleles producing these neuronal phenotypes, expression of the cytoplasmic dynein 8 kDa light chain gene is completely absent. These results demonstrate a new function for the cytoplasmic dynein light chain in the regulation of axonogenesis and may provide a point of entry for studies of the role of cellular motors in growth cone guidance (Phillis, 1996).
Intrinsically disordered protein (IDP) duplexes composed of two IDP chains cross-linked by bivalent partner proteins form scaffolds for assembly of multiprotein complexes. The N-terminal domain of dynein intermediate chain (N-IC) is one such IDP that forms a bivalent scaffold with multiple dynein light chains including LC8, a hub protein that promotes duplex formation of diverse IDP partners. N-IC also binds a subunit of the dynein regulator, dynactin. This study characterized interactions of a yeast ortholog of N-IC (N-Pac11) with yeast LC8 (Dyn2) or with the intermediate chain-binding subunit of yeast dynactin (Nip100). Residue level changes in Pac11 structure are monitored by NMR spectroscopy, and binding energetics are monitored by isothermal titration calorimetry (ITC). N-Pac11 is monomeric and primarily disordered except for a single alpha-helix (SAH) at the N terminus and a short nascent helix, LH, flanked by the two Dyn2 recognition motifs. Upon binding Dyn2, the only Pac11 residues making direct protein-protein interactions are in and immediately flanking the recognition motifs. Dyn2 binding also orders LH residues of Pac11. Upon binding Nip100, only Pac11 SAH residues make direct protein-protein interactions, but LH residues at a distant sequence position and L1 residues in an adjacent linker are also ordered. The long distance, ligand-dependent ordering of residues reveals new elements of dynamic structure within IDP linker regions (Jie, 2015).
The cytoplasmic dynein motor generates pulling forces to center and orient the mitotic spindle within the cell. During this positioning process, dynein oscillates from one pole of the cell cortex to the other but only accumulates at the pole farthest from the spindle. This study shows that dynein light chain 1 (DYNLL1) is required for this asymmetric cortical localization of dynein and has a specific function defining spindle orientation. DYNLL1 interacted with a spindle-microtubule-associated adaptor formed by CHICA and HMMR via TQT motifs in CHICA. In cells depleted of CHICA or HMMR, the mitotic spindle failed to orient correctly in relation to the growth surface. Furthermore, CHICA TQT motif mutants localized to the mitotic spindle but failed to recruit DYNLL1 to spindle microtubules and did not correct the spindle orientation or dynein localization defects. These findings support a model where DYNLL1 and CHICA-HMMR form part of the regulatory system feeding back spindle position to dynein at the cell cortex (Dunsch, 2012).
Cytoplasmic dynein is a large multisubunit complex involved in retrograde transport and the positioning of various organelles. Dynein light chain (LC) subunits are conserved across species; however, the molecular contribution of LCs to dynein function remains controversial. One model suggests that LCs act as cargo-binding scaffolds. Alternatively, LCs are proposed to stabilize the intermediate chains (ICs) of the dynein complex. To examine the role of LCs in dynein function, Saccharomyces cerevisiae, in which the sole function of dynein is to position the spindle during mitosis, was used. This study reports that the LC8 homologue, Dyn2, localizes with the dynein complex at microtubule ends and interacts directly with the yeast IC, Pac11. Two Dyn2-binding sites were identified in Pac11 that exert differential effects on Dyn2-binding and dynein function. Mutations disrupting Dyn2 elicit a partial loss-of-dynein phenotype and impair the recruitment of the dynein activator complex, dynactin. Together these results indicate that the dynein-based function of Dyn2 is via its interaction with the dynein IC and that this interaction is important for the interaction of dynein and dynactin. In addition, these data provide the first direct evidence that LC occupancy in the dynein motor complex is important for function (Stuchell-Brereton, 2011).
Bassoon and the related protein Piccolo are core components of the presynaptic cytomatrix at the active zone of neurotransmitter release. They are transported on Golgi-derived membranous organelles, called Piccolo-Bassoon transport vesicles (PTVs), from the neuronal soma to distal axonal locations, where they participate in assembling new synapses. Despite their net anterograde transport, PTVs move in both directions within the axon. How PTVs are linked to retrograde motors and the functional significance of their bidirectional transport are unclear. This study reports the direct interaction of Bassoon with dynein light chains (DLCs) DLC1 and DLC2, which potentially link PTVs to dynein and myosin V motor complexes. This study demonstrates that Bassoon functions as a cargo adapter for retrograde transport and that disruption of the Bassoon-DLC interactions leads to impaired trafficking of Bassoon in neurons and affects the distribution of Bassoon and Piccolo among synapses. These findings reveal a novel function for Bassoon in trafficking and synaptic delivery of active zone material (Fejtova, 2009).
The operations within a living cell depend on the collective activity of networks of proteins, sometimes termed "interactomes". Within these networks, most proteins interact with few partners, while a small proportion of proteins, called hubs, participate in a large number of interactions and play a central role in organizing these interactomes. LC8 was first discovered as an essential component of the microtubule-based molecular motor dynein and as such is involved in fundamental processes, including retrograde vesicular trafficking, ciliary/flagellar motility, and cell division. More recently, evidence has accumulated that LC8 also interacts with proteins that are not clearly connected with dynein or microtubule-based transport, including some with roles in apoptosis, viral pathogenesis, enzyme regulation, and kidney development. This paper introduces the idea that LC8 is a hub protein essential in diverse protein networks, and its function as a dynein light chain is but one of many. It is further proposed that the crucial regulatory roles of LC8 in various systems are due to its ability to promote dimerization of partially disordered proteins (Barbar, 2008).
A 10 kDa dynein light chain (DLC), previously identified as a tail light chain of myosin Va, may function as a cargo-binding and/or regulatory subunit of both myosin and dynein. This study identified and characterized the binding site of DLC on myosin Va. Fragments of the human myosin Va tail and the DLC2 isoform were expressed, and their complex formation was analyzed by pull-down assays, gel filtration, and spectroscopic methods. DLC2 was found to bind as a homodimer to a approximately 15 residue segment (Ile1280-Ile1294) localized between the medial and distal coiled-coil domains of the tail. The binding region contains the three residues coded by the alternatively spliced exon B (Asp1284-Lys1286). Removal of exon B eliminates DLC2 binding. Co-localization experiments in a transfected mammalian cell line confirm the finding that exon B is essential for DLC2 binding. Using circular dichroism, it was demonstrated that binding of DLC2 to a approximately 85 residue disordered domain (Pro1235-Arg1320) induces some helical structure and stabilizes both flanking coiled-coil domains (melting temperature increases by approximately 7 degrees C). This result shows that DLC2 promotes the assembly of the coiled-coil domains of myosin Va. Nuclear magnetic resonance spectroscopy and docking simulations show that a 15 residue peptide (Ile1280-Ile1294) binds to the surface grooves on DLC2 similarly to other known binding partners of DLCs. When these data are taken together, they suggest that exon B and its associated DLC2 have a significant effect on the structure of parts of the coiled-coil tail domains and such a way could influence the regulation and cargo-binding function of myosin Va (Hodi, 2006).
Search PubMed for articles about Drosophila Cut up
Barbar, E. (2008). Dynein light chain LC8 is a dimerization hub essential in diverse protein networks. Biochemistry 47(2): 503-508. PubMed ID: 18092820
Barron, D.A. and Moberg, K. (2016). Inverse regulation of two classic Hippo pathway target genes in Drosophila by the dimerization hub protein Ctp. Sci Rep 6: 22726. PubMed ID: 26972460
Batlevi, Y., Martin, D. N., Pandey, U. B., Simon, C. R., Powers, C. M., Taylor, J. P. and Baehrecke, E. H. (2010). Dynein light chain 1 is required for autophagy, protein clearance, and cell death in Drosophila. Proc Natl Acad Sci U S A 107(2): 742-747. PubMed ID: 20080745
Dick, T., Ray, K., Salz, H. K. and Chia, W. (1996). Cytoplasmic dynein (ddlc1) mutations cause morphogenetic defects and apoptotic cell death in Drosophila melanogaster. Mol Cell Biol 16(5): 1966-1977. PubMed ID: 8628263
Dunsch, A. K., Hammond, D., Lloyd, J., Schermelleh, L., Gruneberg, U. and Barr, F. A. (2012). Dynein light chain 1 and a spindle-associated adaptor promote dynein asymmetry and spindle orientation. J Cell Biol 198(6): 1039-1054. PubMed ID: 22965910
Fejtova, A., Davydova, D., Bischof, F., Lazarevic, V., Altrock, W. D., Romorini, S., Schone, C., Zuschratter, W., Kreutz, M. R., Garner, C. C., Ziv, N. E. and Gundelfinger, E. D. (2009). Dynein light chain regulates axonal trafficking and synaptic levels of Bassoon. J Cell Biol 185(2): 341-355. PubMed ID: 19380881
Hodi, Z., Nemeth, A. L., Radnai, L., Hetenyi, C., Schlett, K., Bodor, A., Perczel, A. and Nyitray, L. (2006). Alternatively spliced exon B of myosin Va is essential for binding the tail-associated light chain shared by dynein. Biochemistry 45(41): 12582-12595. PubMed ID: 17029413
Jie, J., Lohr, F. and Barbar, E. (2015). Interactions of Yeast Dynein with Dynein Light Chain and Dynactin: GENERAL IMPLICATIONS FOR INTRINSICALLY DISORDERED DUPLEX SCAFFOLDS IN MULTIPROTEIN ASSEMBLIES. J Biol Chem 290(39): 23863-23874. PubMed ID: 26253171
Joti, P., Ghosh-Roy, A. and Ray, K. (2011). Dynein light chain 1 functions in somatic cyst cells regulate spermatogonial divisions in Drosophila. Sci Rep 1: 173. PubMed ID: 22355688
Jurado, S., Conlan, L. A., Baker, E. K., Ng, J. L., Tenis, N., Hoch, N. C., Gleeson, K., Smeets, M., Izon, D. and Heierhorst, J. (2012a). ATM substrate Chk2-interacting Zn2+ finger (ASCIZ) is a bi-functional transcriptional activator and feedback sensor in the regulation of dynein light chain (DYNLL1) expression. J Biol Chem 287(5): 3156-3164. PubMed ID: 22167198
Jurado, S., Gleeson, K., O'Donnell, K., Izon, D. J., Walkley, C. R., Strasser, A., Tarlinton, D. M. and Heierhorst, J. (2012b). The Zinc-finger protein ASCIZ regulates B cell development via DYNLL1 and Bim. J Exp Med 209(9): 1629-1639. PubMed ID: 22891272
King, S. M. (2008). Dynein-independent functions of DYNLL1/LC8: redox state sensing and transcriptional control. Sci Signal 1(47): pe51. PubMed ID: 19036713
Kreko-Pierce, T. and Eaton, B. A. (2017). The Drosophila LC8 homolog cut up specifies the axonal transport of proteasomes. J Cell Sci 130(19): 3388-3398. PubMed ID: 28808087
Murphey, R. K., Caruccio, P. C., Getzinger, M., Westgate, P. J. and Phillis, R. W. (1999). Dynein-dynactin function and sensory axon growth during Drosophila metamorphosis: A role for retrograde motors. Dev Biol 209(1): 86-97. PubMed ID: 10208745
Navarro, C., Puthalakath, H., Adams, J. M., Strasser, A. and Lehmann, R. (2004). Egalitarian binds dynein light chain to establish oocyte polarity and maintain oocyte fate. Nat Cell Biol 6(5): 427-435. PubMed ID: 15077115
Phillis, R., Statton, D., Caruccio, P. and Murphey, R. K. (1996). Mutations in the 8 kDa dynein light chain gene disrupt sensory axon projections in the Drosophila imaginal CNS. Development 122(10): 2955-2963. PubMed ID: 8898210
Rapali, P., Radnai, L., Suveges, D., Harmat, V., Tolgyesi, F., Wahlgren, W. Y., Katona, G., Nyitray, L. and Pal, G. (2011a). Directed evolution reveals the binding motif preference of the LC8/DYNLL hub protein and predicts large numbers of novel binders in the human proteome. PLoS One 6(4): e18818. PubMed ID: 21533121
Rapali P., Szenes A., Radnai L., Bakos A., Pal G., et al. (2011b). DYNLL/LC8: a light chain subunit of the dynein motor complex and beyond. FEBS J. 278: 2980-2996. PubMed ID: 21777386
Sengupta, S., Rath, U., Yao, C., Zavortink, M., Wang, C., Girton, J., Johansen, K. M. and Johansen, J. (2016). Digitor/dASCIZ has multiple roles in Drosophila development. PLoS One 11(11): e0166829. PubMed ID: 27861562
Slevin, L. K., Romes, E. M., Dandulakis, M. G. and Slep, K. C. (2014). The mechanism of dynein light chain LC8-mediated oligomerization of the Ana2 centriole duplication factor. J Biol Chem 289(30): 20727-20739. PubMed ID: 24920673
Stevens, N. R., Dobbelaere, J., Brunk, K., Franz, A. and Raff, J. W. (2010). Drosophila Ana2 is a conserved centriole duplication factor. J Cell Biol 188(3): 313-323. PubMed ID: 20123993
Stuchell-Brereton, M. D., Siglin, A., Li, J., Moore, J. K., Ahmed, S., Williams, J. C. and Cooper, J. A. (2011). Functional interaction between dynein light chain and intermediate chain is required for mitotic spindle positioning. Mol Biol Cell 22(15): 2690-2701. PubMed ID: 21633107
Wang, C., Li, S., Januschke, J., Rossi, F., Izumi, Y., Garcia-Alvarez, G., Gwee, S. S., Soon, S. B., Sidhu, H. K., Yu, F., Matsuzaki, F., Gonzalez, C. and Wang, H. (2011). An ana2/ctp/mud complex regulates spindle orientation in Drosophila neuroblasts. Dev Cell 21(3): 520-533. PubMed ID: 21920316
Zaytseva, O., Tenis, N., Mitchell, N., Kanno, S., Yasui, A., Heierhorst, J. and Quinn, L. M. (2014). The novel zinc finger protein dASCIZ regulates mitosis in Drosophila via an essential role in dynein light-chain expression. Genetics 196(2): 443-453. PubMed ID: 24336747
date revised: 15 February 2018
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